Hepatitis C virus (HCV) is one of the most widespread infectious diseases in the world. About 170 million people are infected with HCV worldwide with a yearly incidence of 3-4 million. While the acute phase of infection is mostly asymptomatic, the majority of acutely infected individuals develops chronic hepatitis and is at increased risk of developing liver cirrhosis and hepatocellular carcinoma. Thus, HCV infection is a major contributor to end-stage liver disease and in developed countries to liver transplantation.
HCV is a small, enveloped virus classified as a member of the Flaviviridae family. Its genome consists of a 9.6 kb single stranded RNA of positive polarity composed of 5′ and 3′ untranslated regions (UTR) and one long open reading frame (ORF) encoding a polyprotein, which is co- and posttranslationally cleaved and thus yields the structural (Core, E1, E2), p7 and nonstructural (NS2, NS3, NS4A, NS4B, NS5A, NS5B) proteins.
HCV isolates from around the world exhibit significant genetic heterogeneity. At least 6 major HCV genotypes (genotypes 1-6) have been identified, which differ by 31-33% at the nucleotide level. In addition, there are numerous subtypes (a, b, c, etc.). In general different subtypes and isolates differ respectively by 20-25% and 2-8% at the nucleotide level. In the U.S., the majority of HCV infected individuals has genotype 1 (1a or 1b), while most others are infected with genotype 2 (2a or 2b) or 3a. Genotype 3a is more prevalent in Europe infecting up to 50% of patients in several countries with a high prevalence in specific risk groups, such as intravenous drug users and its prevalence in Europe is expected to rise.
Furthermore, genotype 3a is very prevalent in many highly populated countries in Asia such as India and Pakistan, as well as the former USSR, Australia and Brazil. In HCV infected patients, genotype 3 was found to be associated with more pronounced hepatic steatosis compared to other genotypes. The only approved therapy for HCV, combination therapy with interferon and ribavirin, is expensive and associated with severe side effects and contraindications. Sustained viral response can be achieved in only about 55% of treated patients in general, in 85-90% of patients infected with genotypes 2 and 3 and only in 40-50% of patients infected with genotype 1. There is no vaccine against HCV.
Since its discovery in 1989, research on HCV has been hampered by the lack of appropriate cell culture systems allowing for research on the complete viral life cycle as well as new therapeutics and vaccines. Full-length consensus cDNA clones of HCV strain H77 (genotype 1a) and J6 (genotype 2a) shown to be infectious in the chimpanzee model, were apparently not infectious in vitro. Replicon systems permitted the study of HCV RNA replication in cell culture using the human liver hepatoma cell line Huh7 but were dependent on adaptive mutations that were deleterious for infectivity in vivo.
In 2001, a genotype 2a isolate (JFH1) was described (Kato et al., 2001), which yielded high RNA titers in the replicon system without adaptive mutations (Kato et al., 2003).
A major breakthrough occurred in 2005, when formation of infectious viral particles was reported after transfection of RNA transcripts from the JFH1 full-length consensus cDNA clone into Huh7 cells and the derived Huh7.5.1 (Wakita et al., 2005) (Zhong et al., 2005).
At the same time, Lindenbach et al. demonstrated that the intragenotypic 2a/2a recombinant genome (J6/JFH1), in which the structural genes (C, E1, E2), p7 and NS2 of JFH1 were replaced by the corresponding genes of the infectious cDNA clone J6CF, produced infectious viral particles in Huh7.5 cells (a cell line derived from bulk Huh7 cells) with an accelerated kinetic (Lindenbach et al., 2005). Cell culture derived J6/JFH viruses were apparently fully viable in vivo (Lindenbach et al., 2006). Intragenotypic and intergenotypic recombinant HCV genomes are naturally occurring. Interestingly, in several of these isolates the recombination breakpoint apparently maps in close proximity to the NS2/NS3 junction, the site of recombination in the J6/JFH genomes.
Despite the importance of the described cell culture systems they represent only a single subtype (genotype 2a) of HCV.
Pietschmann et al. 2006 disclose the construction and characterization of infectious intragenotypic and intergenotypic hepatitis C virus recombinants. The authors created a series of recombinant genomes allowing production of infectious viral particles containing Core through NS2 of genotype 1a, 1b, 2a and 3a by constructing intra- and intergenotypic recombinant genomes between the JFH1 isolate and the HCV isolates: H77 (genotype 1a), Con1 (genotype 1b), J6 (genotype 2a) and 452 (genotype 3a) respectively. Thus, disclosing a genotype 3a isolate completely different from the isolate disclosed in the present application.
The infectious titers of the 1a, 1b and 3a genotypes disclosed in Pietschmann et al. 2006 are not at a level sufficiently high for practical utilization in functional analysis, drug and vaccine development or most other applications. For such applications, including screening of potential drugs and development of potential vaccine candidates the skilled person will know that infectivity titers below 103 TCID50/mL contain insufficient amounts of infectious virus. Besides from disclosing a genotype 3a isolate different from the genotype 3a isolate disclosed in the present application Pietschmann et al. 2006 provides no sequence data of the virus produced in the cell culture. Accordingly, the study does not attempt cell culture adaptation of the genotype recombinants, e.g. by serial passage of cell culture derived viruses to naïve cells and it is not investigated whether adaptive mutations developed after transfection in cell culture.
In fact, Pietschmann et al does not even provide any sequence data of the virus produced in the cell culture.